Opioids such as morphine are powerful analgesics that are widely used in the management of pain. Opioid receptors are prone to desensitization and, in some cases, down regulation following agonist treatment, and these processes ultimately lead to the development of tolerance rendering continuation of opioid therapy less effective. Receptor activities are largely governed by post-translational modifications, especially phosphorylation, and interactions with scaffolding and signaling proteins that modulate and cooperate in receptor signal transduction. Our lab has developed HEK 293 cells stably transfected with the rat kappa opioid receptor (rKOR) with a FLAG epitope at the amino terminus. It is our hypothesis that we can investigate post-translational receptor modifications and identify receptor-associated proteins by mass spectrometric analysis of the purified rKOR. To purify rKOR, wheat germ agglutinin chromatography followed by two rounds of anti-FLAG immunoaffinity chromatography and SDS-PAGE will be used. Following purification and enzymatic and chemical digestion, rKOR will be analyzed by matrix assisted laser desorption ionization (MALDI) tandem mass spectrometry to identify post-translational modifications, with particular interest in identifying sites of phosphorylation, palmitoylation and ubiquitination. In addition, proteins that co-immunoprecipitate with rKOR will be identified using mass spectrometry. To confirm these protein-protein interactions, western blotting of anti-FLAG immunoprecipitations from rKOR and untransfected HEK 293 cells (negative control) will be done. By utilizing these techniques to learn more about the molecular mechanisms of kappa opioid receptor regulation, much can be gained to further the utility of this receptor as a target for diuretics, antipruritics, anticonvulsives, and alleviation of craving in cocaine addicts. Prolonged treatment of opioid drugs leads to the development of tolerance rendering these drugs less effective. Examining the mechanisms by which the kappa opioid receptor is regulated may lead to improved utilization of this receptor for the treatment of pain, epilepsy, itching, and cocaine addiction. [unreadable] [unreadable] [unreadable]